T. versicolor was previously identified from decomposing wood samples obtained from the field , and in the present study, its genotype was re-confirmed by PCR analysis, using ITS1-F and ITS4 primers. Sequenced PCR products (Supplementary Table 1) were aligned with available sequences in the NCBI database  and were found to have up to 99% identity with T. versicolor (FJ810146.1). Therefore, T. versicolor (CBS 109428) from the CBS Culture Collection (The Netherlands) was used in all the experiments described in this paper.
T. versicolor Thrives in Media Containing Hexoses and Xylose Under Hypoxic Conditions
Using their extra-cellular enzymatic complexes basidiomycetous white rot fungi, such as T. versicolor, are responsible for extensive biodegradation of lignin in nature [15, 16]. The most rapid and extensive degradation caused by certain fungi, particularly white rot fungi, occurs in highly aerobic environments . In the growth experiments presented here, with differing sugar combinations, T. versicolor grew exponentially, producing biomasses of 12.6 g/l FW and 7.2 g/l FW after 66 h in MBMC and XY11 media (Table 1), respectively, under hypoxic conditions (Fig. 1b). Furthermore, T. versicolor growth was stable in both media for up to 354 h (Fig. 1b). However, under anoxic conditions T. versicolor did not grow during the 354 h incubation period in either medium, in accordance with results of a previous study in which liquid culture bottle headspaces of T. versicolor cultures were flushed daily with 95% replacement of the atmospheric gases to promote typical growth .
In addition, growth behaviour in both media showed a typical pattern, the total biomass produced during the 354 h under hypoxic conditions being significantly higher in MBMC than in XY11 medium (Fig. 1b). The ability of T. versicolor to grow rapidly using various carbohydrate sources, including hexoses (glucose and mannose) and pentoses (xylose) explains its ability to thrive in diverse ecosystems. Moreover T. versicolor produces generative filamentous hyphae (Fig. 1a), like most Trametes species, with three types of structure: skeletal, generative and binding [18, 19], making it easy to handle. The ease of handling, stability, versatility and rapid multiplication of T. versicolor offer substantial advantages for its use in industrial bio-refineries . Hence, there is intensive research into their use not only in bio-fuel generation, but also in bioremediation, effluent treatment, biosensors, synthetic chemistry and the pulp and paper, food, cosmetic and textile industries.
P. tannophilus shows enhanced aerobic growth and fermentation of xylose into ethanol after periodic additions of glucose, while similar additions to anaerobic cultures have no effect on its xylose utilization . To determine the effects of varying combinations of glucose and xylose on the growth of T. versicolor, it was cultivated on media with various ratios of sugars (G11XY11, G6XY11 and G2XY11, containing 1:1, 1:1.8 and 1:5.5 glucose/xylose ratios, respectively; Table 1) in both hypoxic and anoxic conditions. The cultures were sampled after 18, 66, 114, 234 and 354 h at 27°C. Biomass (FW) production measurements indicated that growth was rapid for up to 66 h in all three glucose/xylose media (Fig. 3a) and was stable for up to 354 h under hypoxic conditions. However, under anoxic conditions no biomass production was detected. Furthermore, under hypoxic growth conditions, slight differences were detected in the biomass produced over 354 h between the three media (Fig. 3a). Our results support those of previous findings that efficient aeration of Rhizopus oryzae liquid cultures is important for rapid assimilation of hexoses and pentoses .
Studies of genetically engineered xylose-fermenting yeast has earlier shown that glucose utilization proceeds both promotion and inhibition of xylose usage  in mixed sugar fermentation, while aeration doubled the specific glucose-fermentation rate in P. tannophilus cultures . To elucidate the sugar utilizing ability of T. versicolor in hexose and xylose mixtures, the sugars present in MBMC and XY11 media after 18, 66, 114, 234 and 354 h of cultivation at 27°C under hypoxic conditions were analysed. The results clearly showed that T. versicolor could utilize mannose, glucose and xylose simultaneously under hypoxic conditions in MBMC medium for up to 354 h (Fig. 2). This is a unique physiological characteristic of T. versicolor compared with other xylose-utilizing microorganisms, most of which use hexoses at higher rates than pentoses such as xylose in early growth stages [23, 24], while in our experiments T. versicolor utilized mannose, glucose and xylose simultaneously, consuming 32%, 44% and 36% of these sugars, respectively, within 66 h (Fig. 2). In addition, our data indicate that T. versicolor can effectively use xylose as a sole carbohydrate source over 354 h (Fig. 5a), after which ca. 90% of xylose had been utilized. Moreover, T. versicolor utilized 53% of the xylose during the first 18 h of culture (Fig. 5a). This further explains the physiological adaptability that T. versicolor has evolved, from its exposure to the diverse substrates in its environment.
In this study, among the hexoses T. versicolor showed a preference for glucose over mannose with concomitant consumption of xylose during the first 66 h of cultivation (Fig. 2). While, during the later growth period (66–354 h), 58%, 55% and 44% of the mannose, glucose and xylose were used, respectively (Fig. 2). Therefore, our sugar utilization analysis indicates that there was no hexose (glucose or mannose) co-substrate inhibition of xylose use, in contrast to an earlier report on genetically engineered xylose-fermenting S. cerevisiae . S. cerevisiae takes up xylose by diffusion trough non-specific hexose transporters which have lower affinity for xylose than for glucose . Therefore, xylose transport of S. cerevisiae is competitively inhibited by glucose, and xylose is consumed only after depletion of glucose . However, our results strongly indicate that T. versicolor takes up both hexose (glucose and mannose) and pentose (xylose) simultaneously, while xylose is transported by the same system as hexose (Fig. 2). The importance of sugar transporter systems was demonstrated by Hamacher et al. , who showed that deletion of 18 Hxts transporter genes in a genetically modified S. cerevisiae strain (TMB 3201) capable of growth on xylose-based medium caused loss of the yeast’s ability to take up and grow on xylose. In addition, since no hexose co-substrate inhibition was observed, T. versicolor presumably has mechanisms allowing sugar catabolism under hypoxic conditions, including the continuous of supply reactants required for efficient glycolysis and ethanol fermentation (inter alia glucose, which elevates the intracellular metabolic pool of glyceraldehyde-3-phosphate, thus facilitating efficient operation of the pentose phosphate pathway; ).
In most fungi and xylose-fermenting yeasts (e.g. P. stipitis, P. tannophilus and C. shehatae), d-xylose is converted to d-xylulose by two oxidoreductases in reactions involving the co-factors NAD(P)H and NAD(P)+ through the reductase pathway, where d-xylose is initially reduced to xylitol by NAD(P)H-dependent XR [26–29]. Then xylitol is oxidized to d-xylulose by XDH [26, 28, 30, 31]. d-xylulose is finally phosphorylated to xylulose-5-phosphate by XK . To determine the activities of enzymes involved in the xylose catabolism of T. versicolor, XR, XDH and XK activities were analysed in mycelia grown in MBMC, G11XY11, G6XY11 and G2XY11 media (Table 1) after 18, 66, 114, 234 and 354 h of cultivation at 27°C under hypoxic conditions. XR and XDH activities of mycelia grown in MBMC medium increased exponentially for 66 h, reaching 54.7 and 48.3 μmol min−1 g−1 protein, respectively (Fig. 2). These enzymatic activities of XR and XDH after 66 h of cultivation explain the efficient utilization of xylose (Fig. 2). Between 66 and 354 h of cultivation, XR activity decreased slightly and then remained at ca. 39 μmol min−1 g−1 protein, while XDH activity fell slightly and then increased to 59.5 μmol min−1 g−1 protein (Fig. 2). In addition, the XK activity of mycelia grown in MBMC medium increased exponentially, up to 20.6 μmol min−1 g−1 protein after 18 h of cultivation, then decreased slightly to a constant 17–20 μmol min−1 g−1 protein for up to 354 h growth. The correlations between XR, XDH and XK activities from 0 to 354 h and xylose utilization over the same period (Fig. 2) indicate that T. versicolor can efficiently catabolise xylose in hexose and xylose mixtures and that xylose transport is not inhibited by hexoses. Moreover, this is corroborated by XR, XDH and XK activities measured in mixtures containing different glucose/xylose ratios (Fig. 3b). The activities of XR, XDH and XK all increased in every medium up to 66 h, while they subsequently decreased and remained activity up to 354 h (Fig. 3b). No significant differences were found between the XR, XDH and XK activities in mixtures with different glucose/xylose ratios, with identical xylose concentrations (11 g/l) over 354 h (Fig. 3b), confirming that xylose utilization by T. versicolor is not inhibited by glucose in media where, xylose concentrations are equal or higher than glucose. In addition, significant differences were found between the XR, XDH and XK activities (Fig. 2) in MBMC medium, (glucose and mannose represent 6-carbon 36.7 g/l and xylose represents 5-carbon 11 g/l mixture, Table 1) with respect to different glucose/xylose ratios (Fig. 3b) containing identical xylose concentrations (11 g/l) over 354 h, confirming that xylose utilization by T. versicolor is improved by hexoses in MBMC media (Fig. 2).
Ethanol Production by T. versicolor from Hexoses and Xylose
The industrial S. cerevisiae strain MA-R4 reportedly gives high ethanol yields from a non-sulphuric acid hydrolysate of eucalyptus wood chips , and other mixed glucose and xylose carbon sources . Generally, industrial S. cerevisiae strains are superior ethanol producers due to their tolerance of inhibitors and high ethanol productivity under industrial conditions, but to date limited numbers of industrial S. cerevisiae strains have been generated for xylose fermentation [34–39]. In addition, T. versicolor was able to produce 6.94 g/l ethanol from spent sulphite liquor after 36 h of fermentation . To determine the ability of T. versicolor to produce ethanol from hexoses and xylose, the ethanol contents of the MBMC, G11XY11, G6XY11 and G2XY11 media were analysed after 18, 66, 114, 234 and 354 h of cultivation, and XY50 (Table 1) was analysed after 714 h at 27°C, under both anoxic and hypoxic conditions. The ethanol content of the MBMC culture medium gradually increased over 354 h, to a maximum of 20.0 ± 0.79 g/l (Fig. 4a) under hypoxic condition, while under anoxic conditions, ethanol production was not detected, in accordance with reports by  that the growth and ethanol production rates of P. tannophilus strongly depend on aeration. However, for commercial use of T. versicolor, we need to improve the fermentation rate by increasing the amount of initial inoculums and bio technologically improvements in xylose-utilizing pathway.
Furthermore, P. stipitis, the most efficient natural xylose-fermenting yeast known, produced 0.48 g/g ethanol from xylose when cultured continuously with limited oxygen  and ethanol formation from xylose by recombinant S. cerevisie  has also been reported. In our study, T. versicolor produced ethanol in media with various xylose/glucose ratios at increasing rates for up to 66 h, and slightly increasing rates for up to 354 h (Fig. 4b), with final ethanol contents in the media of 9.02 g/l ± 0.05 in (G11XY11), 5.4 g/l ± 0.19 (G6XY11) and 1.71 ± 0.08 (G2XY11). The theoretical yield of ethanol from genetically engineered xylose-fermenting yeast was 0.51 g ethanol/g for glucose and 0.51 g ethanol/g for xylose . Therefore, our data suggest that T. versicolor can utilize xylose for ethanol production for up to 354 h in media with various hexose and xylose mixtures under hypoxic conditions. In G11XY11 medium, the ethanol yield was 80% of the theoretical maximum after 354 h under hypoxic conditions, since the actual yield was 9.02 ± 0.05 compared with the theoretical maximum of 11.22 g/l (0.51 g ethanol/g glucose and 0.51 g ethanol/g xylose). Therefore, our results show that T. versicolor produced ethanol from xylose with favourable efficiency when the glucose/xylose ratio was 1:1, under hypoxic conditions.
In addition, XY50 medium ethanol production was detected after 354 h and increased (2.97 ± 0.2 g/l) at 714 h under hypoxic conditions. Furthermore, in XY50 medium no ethanol production was observed over 714 h under anoxic conditions (Fig. 5b). Previous studies have also shown that fungal-mix including T. versicolor can produce ethanol from mixtures of hexoses and xylose , and that the fungus P. chrysosporium can degrade lignin in biomass to ethanol by fermenting both hexoses and pentoses . Furthermore, utilization of ethanol by T. versicolor may explain its late ethanol production in XY50 medium (2.97 ± 0.2 g/l) under hypoxic conditions (Fig. 5b). This hypothesis is supported by findings that at high aeration rates the ethanol yield of P. tannophilus decreases, since it respires ethanol in the presence of xylose [21, 43].
The activation of ethanol fermentation is required for recycling NAD+ under limited oxygen conditions, which is essential for maintaining glycolysis. To maintain ethanolic fermentation effectively, ADH and PDC activities are important. Therefore, to determine the contributions of key enzymes to ethanol fermentation by T. versicolor, ADH activity was analysed in mycelia grown in MBMC, G11XY11, G6XY11 and G2XY11 media after 18, 66, 114, 234 and 354 h of cultivation at 27°C under hypoxic conditions. Its ADH activity in MBMC media increased continuously during this time (Fig. 4a), reaching 119.5 ± 4.9 μmol min−1 g−1 protein, and was correlated with ethanol production. Furthermore, ADH activity in the xylose and glucose mixtures increased rapidly over 66 h, but declined rapidly after 114 h in all three mixtures (Fig. 4b). Interestingly, although significant differences were recorded in ethanol production among mycelia cultivated in the different xylose and glucose mixtures over 354 h, surprisingly, their ADH activities were similar and showed similar trends (Fig. 4b). Rapid increases in ADH activity during 66 h of cultivation observed in all three glucose and xylose mixtures were correlated with the ethanol production in the respective cultures (Fig. 4b). In addition, over 66 h with all three glucose and xylose mixtures, biomass production (Fig. 3a) was correlated with XR, XDH and XK activities (Fig. 3b), providing further evidence that T. versicolor expresses xylose catabolic pathways as well as producing ethanol during the active growth stage in all hexose and xylose mixtures used here.